Background: Nearly 200,000 female Veterans will be diagnosed with breast cancer during their lifetime representing a significant health problem. Metastatic breast cancer is uniformly fatal. Objective: Our goal is to identify new therapeutic approaches to prevent mortality from breast cancer metastasis. We seek novel approaches to exploit the established contribution of the Cyclooxygenase-2 (COX-2) pathway to breast tumor growth and dissemination. The principle COX-2 product, prostaglandin E2 (PGE2) exerts biological effects by binding to the E series of G protein-coupled prostaglandin receptors EP1-4. Results: We have made significant progress towards elucidating the unique role of individual EP receptors on malignant and host immune cells. EP1 suppresses while EP4 promotes metastasis. Our most important finding is that EP4 supports the survival of breast cancer stem cells (BCSC) with heightened tumorigenic, metastatic and chemotherapy resistant properties that contribute to relapse and mortality. EP4 also subverts innate antitumor immunity by inhibiting Natural Killer (NK) cells and inducing immune suppressive myeloid derived suppressor cells (MDSC) but the role of NK and MDSC in control of BCSC is not known. Our central hypothesis is that EP4 promotes metastasis by directly supporting BCSC and indirectly by suppressing antitumor immunity. Systemic targeting of multiple EP4-positive cells will result in additive, if not synergistic therapeutic responses. BCSC, expressing high levels of EP4 will be inhibited directly by EP4 blockade. EP4 inhibitors will protect anti-tumor NK cells from PGE2-mediated immune suppression and will prevent the induction of MDSC that otherwise directly support BCSC survival and prevent effective anti- tumor immunity. Methods: Using a combination of novel pharmacologic EP inhibitors and genetic approaches in orthotopic, xenograft and patient-derived xenograft models of metastatic breast cancer, we will determine the mechanism by which EP4 promotes and EP1 inhibits BCSC. Specific Aims: (1) Determine the effect of EP4/COX-2 targeting on BCSC. We will determine the mechanism by which EP4 supports BCSC, identify the relevant signaling pathways and will determine how EP4 is regulated in BCSC. (2) Determine the effect of EP4/COX-2 targeting on NK effector functions. Little is known regarding the role of innate immunity in the control of BCSC. We will characterize the role of NK cells in the recognition and killing of BCSC and will determine how EP4 regulates these interactions. (3) Determine the effect of EP4 targeting on MDSC and TAM. We will determine if EP4 blockade neutralizes the deleterious immune suppressive effects of MDSC and tumor-associated macrophages (TAM). We will determine if MDSC directly support BCSC through an EP4-regulated mechanism that is independent of effects on innate or adaptive immunity. We will identify a clinically relevant combination therapy that may target cancer stem cells as well as non-stem cell populations. Impact: We will identify EP4 as a new therapeutic target, expressed on host cells and BCSC responsible for relapse and breast cancer mortality. EP4 targeting will overcome immune suppression leading to effective tumor control. Our results will inform the design of clinical trias under development in collaboration with RaQualia/AskAt to determine if administration of an EP4 antagonist reduces breast cancer mortality in female and male Veterans and in the general population with breast cancer.